WO1992010584A1 - Electrode pourvue d'un revetement polymere auquel est liee une enzyme redox - Google Patents

Electrode pourvue d'un revetement polymere auquel est liee une enzyme redox Download PDF

Info

Publication number
WO1992010584A1
WO1992010584A1 PCT/NL1991/000263 NL9100263W WO9210584A1 WO 1992010584 A1 WO1992010584 A1 WO 1992010584A1 NL 9100263 W NL9100263 W NL 9100263W WO 9210584 A1 WO9210584 A1 WO 9210584A1
Authority
WO
WIPO (PCT)
Prior art keywords
latex
membrane
electrode
polypyrrole
electrode according
Prior art date
Application number
PCT/NL1991/000263
Other languages
English (en)
Inventor
Cornelis Gerardus Josephus Koopal
Richardus Bernardus Maria Schasfoort
Roeland Johannes Maria Nolte
Original Assignee
Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno filed Critical Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno
Priority to DE69104496T priority Critical patent/DE69104496T2/de
Priority to US08/075,509 priority patent/US5422246A/en
Priority to EP92901729A priority patent/EP0561966B1/fr
Publication of WO1992010584A1 publication Critical patent/WO1992010584A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/02—Details
    • A61N1/04—Electrodes
    • A61N1/0404—Electrodes for external use
    • A61N1/0472—Structure-related aspects
    • A61N1/0492—Patch electrodes
    • A61N1/0496—Patch electrodes characterised by using specific chemical compositions, e.g. hydrogel compositions, adhesives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/08—Materials for coatings
    • A61L31/10—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61N—ELECTROTHERAPY; MAGNETOTHERAPY; RADIATION THERAPY; ULTRASOUND THERAPY
    • A61N1/00—Electrotherapy; Circuits therefor
    • A61N1/02—Details
    • A61N1/04—Electrodes
    • A61N1/05—Electrodes for implantation or insertion into the body, e.g. heart electrode
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001—Enzyme electrodes
    • C12Q1/002—Electrode membranes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/817—Enzyme or microbe electrode

Definitions

  • Electrode provided with a polymer coatinp with a redox enzyme bound thereto.
  • the invention relates to an electrode which is provided with a polymer coating having a redox enzyme bound thereto and which electrode, depending on the chosen aim, can be used either in a biosensor for the specific detection of certain substances recognisable to the particular enzyme or in the production of chemicals which can be prepared by the particular enzyme.
  • the invention also relates to biosensors and production installations for chemicals, which contain such an electrode.
  • a biosensor of the type described above is disclosed in Anal. Chem. 1990, £2, pp. 1111-1117• More particularly, this literature reference relates to a platinum-plated carbon electrode, which is provided with the redox enzyme glucose oxidase and a layer of polymerised 1,2-diaminobenzene applied by means of electropolymerisation.
  • glucose sensor of this type based on flavin adenine dinucleotide (FAD) - bound glucose oxidase (GOd) , the glucose to be detected is determined indirectly with the aid of the liberated hydrogen peroxide, as is illustrated by the reaction equations given below:
  • the detection of this hydrogen peroxide which is detected at the anode, has the disadvantage that said detection must be carried out under high voltages applied to the sensor, which can give rise to interference by other substances.
  • the hydrogen peroxide has a degrading action on the glucose oxidase used as redox enzyme (see p. 1112, left hand column, paragraph three).
  • an immobilisation of the glucose oxidase preferably takes place using a carbodiimide as agent providing covalent bonds and/or glutaraldehyde as crosslinking agent, which makes the production of such a biosensor fairly labo ⁇ rious.
  • a carbodiimide as agent providing covalent bonds and/or glutaraldehyde as crosslinking agent
  • Sensors and Actuators Bl (1990), pp. 537-541 discloses the use of a polypyrrole film applied to an electrode surface, which film is bonded covalently, via a carbodiimide activation, with redox enzymes such as glucose oxidase and the like. Biosensors having a relatively short response time are obtained in this way. However, in the production of such biosensors a carbodiimide is required as reagent, the use of which is found to be undesirable.
  • Biotechnol. Bioeng. (1988), 31, 6, 3/2, 553 ⁇ 558 discloses an electrocatalysis reactor in which glucose oxidase is used as redox enzyme.
  • This glucose oxidase is applied to the surface of carbon felt by means of carrying out an interface oxidation of the carbon in order to form carboxylic acid groups and an activation of these groups by means of a carbodiimide, followed by immobilisation of the glucose oxidase.
  • a solution of glucose (as starting material for gluconic acid) and benzoquinone (as electron transfer mediator) was passed through the reactor described in this literature reference.
  • the reactor produces gluconic acid from glucose at a rate of about 100 g/hour.litre of reactor.
  • the use of the mediator is also found to be not very suitable.
  • US-A- 4,582,575 relates to electrically conductive composites comprising a dielectric porous substance e.g. fiberglass fabric, and the electrically conducting polymer polypyrrole deposited in the pores and interstices of such substance.
  • a dielectric porous substance e.g. fiberglass fabric
  • the electrically conducting polymer polypyrrole deposited in the pores and interstices of such substance.
  • Such type of composites which pores and interstices have been impregnated i.e. practically filled with polypyrrole do have - considering the high britteness of polypyrrole per se - both a good electrical conductivity and good mechanical properties i.e. are essentially non-brittle and readily handleable.
  • the aim of the invention is to develop an electrode of the type formulated in the preamble, which electrode can be produced in a simple manner and has extensive application possibilities, such as, for example, in a biosensor or in a production installation for the preparation of specific chemicals.
  • a production installation of this type has not yet been described in the literature. It has been found that the abovementioned aim can be achieved with the aid of an electrode which is composed of a membrane, provided with open pores running through said membrane, the walls of the pores having an electrically conducting polymer coating, which polymer coating present on the wall of the open pores contains a redox enzyme bound thereto.
  • one side of the membrane is provided with a conducting layer consisting of for instance a metal or carbon, which layer is in contact with the polymer coating.
  • a significant advantage of the subject of the invention is that in this case no auxiliaries known from the prior art, such as carbodiimides, glutaraldehyde and the like, are needed for immobilisation of the redox enzyme.
  • the redox enzymes present in the pores of the membrane have a better opportunity for retaining their tertiary structure and, on the other hand, in the pores of the membrane are better screened against external influences, such as shear forces exerted thereon and the influences arising when handling the sensor.
  • the membranes used can be many commercially available inert membranes, such as the commercial products Nuclepore membranes, Cyclopore membranes, Anopore membranes and Millipore membranes. More parti ⁇ cularly, Nuclepore membranes are, for example, polycarbonate or polyester membranes, which membranes are provided with uniform cylindrical pores which pass through the membrane. Like the organic Nuclepore membranes, the organic Cyclopore membranes and the in ⁇ organic Anopore membranes also possess pores passing through the membrane. With regard to the thickness of the membranes, it can be stated that this is not critical per se and is usually in the range of 1-20 ⁇ m and advantageously about 10 ⁇ m.
  • the diameter of the pores present in the membranes can vary within wide limits and is usually in the range of 100-10,000 nm (0.1-10 ⁇ m), advantageously in the range of 100-1000 nm.
  • the pore density (number of pores/cm 2 ) is partly dependent on the pore diameter, but is usually in the range of 10 5 -3 x 10 8 .
  • the latex particles - applicable according to the invention - have a diameter in the range of 50-1000 nm, preferably 50-300 nm.
  • suitable latex materials are a.o. (monodispers) polystyrene latex, polymethyl methacrylate latex, a silica latex or a latex of a conducting polymer like polypyrrole and polyacetylene.
  • Specific latex examples are Unisphere latex particles having a diameter of 0 nm (type 10) or 100 nm (type 11)
  • the electrically conducting polymers used can be the polymers known from the prior art, such as the polymers which are based on pyrrole, substituted pyrrole derivatives, thiophene, substituted thiophene derivatives, aniline and substituted aniline derivatives.
  • pyrrole is used as the monomer for the production of an electrically conducting polypyrrole coating.
  • the thickness of the layer applied in the pores partly depends on the diameter of the pores in the membrane, as said pores must still be open following the application of the polymer coating.
  • polypyrrole coatings having a thickness of 50-200 nm are used, but coatings having a thickness deviating therefrom can also be used.
  • redox enzymes for instance oxidases and dehydrogenases
  • redox enzymes may be mentioned as redox enzymes to be applied in the coated pores of the membrane.
  • examples of such enzymes are glucose oxidase, lactose oxidase, galactose oxidase, enoate reductase, hydrogenase and choline dehydrogenase.
  • Glucose oxidase is advantageously used.
  • This redox enzyme is usually present in an amount of 0.02-0.2 U/cm 2 of membrane surface.
  • the electrode according to the invention may be provided on at least one side with a conducting layer consisting of for instance a metal or carbon, which layer can connect the electrode to the measuring instrument coupled thereto.
  • a conducting layer consisting of for instance a metal or carbon
  • suitable metals for this purpose are, inter alia, platinum, gold and palladium, platinum being preferred.
  • the application of the metal layer can be carried out in a known manner, such as, for example, by means of sputtering, vapour deposition and the like.
  • the thickness of such a metal layer is usually 100-500 nm.
  • the electrodes can be produced as follows.
  • the membranes used can be, for example, Nuclepore membranes containing pores having, for example, a diameter of 100-10,000 nm, advantageously 200-8000 nm and preferably 800-1000 nm.
  • the electrically conducting polymer coating can be applied to the pores of the membrane with the aid of an oxidising chemical polymerisation.
  • a pyrrole solution in water for example 0.3-0.8 M pyrrole
  • an iron(III) chloride solution in water (1.5-2. M) are allowed to come into contact with one another, one reagent being placed on one side of the membrane and the other reagent on the other side of the membrane.
  • the pyrrole monomers and the oxidising iron(III) chloride solution meet one another in the pores of the membrane, which results in a polymerisation of the pyrrole.
  • the polymerisation time is not critical and can be, for example, 2-10 min. In this context it is pointed out that the porosity is the main parameter.
  • a polymerisation time appreciably longer than 10 min. leads to non- porous membranes, which are not usable within the framework of the invention.
  • a scanning electron micrograph of a track-etch Cyclopore membrane which pores are coated with the conducting polymer polypyrrole is shown in Fig. 2a.
  • a metal layer of platinum or a similar metal may be applied to one side of the membrane by means of, for example, sputtering using an Edwards Sputtercoater S150B.
  • the membrane is provided in the pores, the walls of which are coated with an electrically conducting polymer, which a redox enzyme such as glucose oxidase by treating the membrane with a redox enzyme-containing solution, with stirring, at a temperature of, for example, 2-10°C, advantageously 4 * C, for at least 0.1 hour, preferably at least 0.5 hour.
  • concentration of glucose oxidase in the solution can vary within wide limits and is about 5 mg/ml.
  • the prepared membranes can be dried overnight in a desiccator over CaCl 2 .
  • latex membranes may be manufactured by casting latex particles on a freshly sputtered metal (e.g. platinum) surface from an aqueous solution, containing the suspended latex particles and - if desired - agarose. Agarose may be added for better attachment of the latex spheres to each other and to the electrode. Subsequently the latex droplet is dried at low temperature to get a uniform layer without cracks in the surface. After drying, the latex modified electrodes are heat-treated resulting in a membrane consisting of a very strong layer of uniform latex particles.
  • a freshly sputtered metal e.g. platinum
  • the amount of agarose present in the latex suspension may be varied to minimize the amount of agarose necessary to yield good latex layers.
  • latex layers were cast from solutions containing 0.125, 0.100, 0.075. and 0.050 wtj. agarose respectively.
  • the agarose content of 0.125 % led to latex layers which were less accessable for polypyrrole coating.
  • Biosensors constructed from latex layers containing this amount of agarose displayed lower activity and very long response times (vide infra) .
  • Lower amounts than 0.125 wtjU of agarose resulted in strong latex layers which could successivefully be treated with polypyrrole.
  • the lowest agarose content tested (0.050 vt%) still yielded strong adhering latex layers. Therefore, in the further synthesis of latex layers, latex suspensions with 0.05 wt% agarose were used.
  • Example 2 thick layers (ca. 5 um) as well as thin layers (ca. 1 ⁇ m) were made. This was accomplished by using two different latex concentrations in the droplet that was cast on the electrode. The droplet size was kept constant. In both cases strong and smooth layers were obtained. Uniform spheres of two different dimensions were used to make latex membranes. Both o particle sizes (112 and 220 nm diameter respectively) yielded layers with interspherical pores in the order of 50-200 nm.
  • the latex layers were modified with polypyrrole by means of electrochemical polymerization.
  • polymerization media having a pyrrole concentration of 0.2-0.8 Molar may be used.
  • the polymerization medium was phosphate buffered saline (PBS) , containing 0.3 M pyrrole. This medium was chosen because enzyme treatment of the polypyrrole modified latex must preferably be performed in PBS. By using the same medium during polymerization and enzyme treatment it was avoided that exchange of dopant ions in the polymer with the solution could take place. When this would happen, major changes in the conducting polymer properties could occur.
  • the electrochemical polymerization was galvanostatically controlled.
  • the amount of charge (current*time) in the polymerization reaction was varied from 100 to 1000 mC/cm 2 .
  • the blackening of the originally white latex layers was proportional to the amount of charge passed. Although only qualitatively, this was a good indication of the proper galvanostatic polymerization of pyrrole in the matrix of latex particles.
  • Scanning electron microscopy (SEM) was used to image the coated latex layers. The images show how the membrane structure changes with polymerisation time (Fig. 2b-d) . In Figure 2b a very open structure is visible between the spheres that make up the bare latex layer.
  • the latex particles are coated with a thin layer of polypyrrole (amount of charge passed is 300 mC/cm 2 ) .
  • the porous structure is still present, while the internal surface now consists largely of polypyrrole.
  • two uncoated latex spheres are visible at the surface.
  • the porosity of the composite layer is lost ( Figure 2d) . Shown in Figure 2d is a thick latex layer, treated for 75 s. (1500 mC/cm 2 ). No enzyme electrodes could be made with these nonporous latices.
  • Fig. 1 Electron shuttle showing the path of the electrons involved in the enzymatic glucose oxidation.
  • Fig. 3s Set-up for carrying out the pyrrole polymerisation in the pores of a membrane:
  • Fig. 4 Plot of GOd activity of polypyrrole-modified Nuclepore membrane, the polypyrrole being provided with glucose oxidase.
  • the time in minutes is plotted on the X axis and the current strength in microamperes on the Y axis.
  • (1) introduction of the polypyrrole-modified Nuclepore membrane (pore diameter: 800 nm; porosity: 3 * 10 7 pores/cm 2 ; material: polyester) obtained in accor ⁇ dance with the example below, the polypyrrole being provided with glucose oxidase, into an electro- chemical cell, as described below for the test for determination of the enzyme activity; (2) : removal of the membrane from the electrochemical cell; (3): introduction of the membrane into the elec- trochemical cell;
  • Fig. 5 Plot of the amperometric response with respect to glucose of a sensor membrane under an argon atmosphere.
  • the time in minutes is plotted on the X axis and the current strength in microamperes on the Y axis.
  • Fig. 6 Plot of the calibration curve for glucose.
  • glucose concentration in mM is plotted on the X axis and the current strength in microamperes on the Y axis.
  • the curve shows the response under argon following injection of 15 ⁇ l, 30 ⁇ l, ⁇ l, 75 ⁇ l. 150 ⁇ l,
  • Fig. 1 Rotating disk electrode assay of glucose oxidase immobilized on 0.112 ⁇ m latex particles. (1) (3): introduction of latex membrane electrode; (2) & (4): withdrawal of latex membrane electrode; (5): introduction of 0.125 U GOd.
  • Fig- 8 Set up for continuous flow measurements of latex membrane biosensors, (a) carrier solution (PBS); (b) sample solution (glucose); (c) peristaltic pump; (d) flow cell; (e) waste; (f) potentiostat; (g) computer; (h) recorder.
  • PBS carrier solution
  • glucose glucose
  • peristaltic pump peristaltic pump
  • Fig. 9 Current response of thin latex layers (ca. 1 ⁇ m) . Latex particle size 0.22 ⁇ m. Measured at 0.35 vs. Ag/AgCl under argon atmosphere with 25 U/ml catalase. Membranes treated with various amounts of pyrrole polymerization charge. (+) 300 mC/cm 2 ; (o) 400 mC/cm 2 .
  • Fig.10 Calibration curves for two latex glucose sensors, made under the same experimental conditions.
  • Latex layer (ca. 1 ⁇ m) consisting of 0.22 ⁇ m particles. Measured at 0.35 vs. Ag/AgCl under argon atmosphere with 25 U/ml catalase.
  • Fig.11 Current response of thick latex layers (ca. 5 ⁇ m) .
  • Fig.12 Calibration curves for optimized latex-polypyrrole sensors. Measured at 0.35 V vs. Ag/AgCl under argon atmosphere with 25 U/ml catalase.
  • Fig.13 Stability of latex-polypyrrole biosensor at continuous operation under ambient atmosphere in the presence of 1 mM glucose. The activity was measured daily by introducing an additional amount of glucose up to a concentration of 5 mM.
  • Fig.l4 Current response of a latex-polypyrrole biosensor upon the addition of glucose. Measurement at 0.35 V vs. Ag/AgCl. Dashed line: in air saturated solution under ambient atmosphere; solid line: in argon flushed solution under argon atmosphere with 25 U/ml catalase.
  • Fig.15 Current response versus time of a latex membrane consisting of 220 nm particles with 100 mC polypyrrole. Measuring potential 0.10 V vs. Ag/AgCl.
  • EXAMPLE I A) Production of an electrode according to the invention. (1) Reagents used.
  • Glucose oxidase (E.C. 1.1.3-4) from Asoergillus niger. type II (25,000 U/g) , and catalase (E.C. 1.11.1.6) from bovine liver, 2800 U/mg were obtained from Sigma.
  • Benzoquinone was obtained from Aldrich (France) and was sublimed before use.
  • Pyrrole was obtained from Merck and anhydrous iron(III) chloride (98%) was obtained from Fluka and these compounds were used in the form supplied.
  • the Nuclepore membranes were obtained from Ankersmit (The Netherlands). All other reagents used were of p.a. grade
  • a polypyrrole-modified filter membrane was pressed against the cooling plate of an Edwards S150B sputtercoater. 100-400 nm of Pt were then applied by sputtering under an argon pressure of 8 mBar and using a sputtering current of 50 mA. The layer thickness was measured using an Edwards FTM5 unit.
  • the membranes which were obtained in accordance with (3) and had an original pore diameter of 800 and 1000 nm were used.
  • a membrane was introduced into 4 ml of a 5 mg/ml GOd solution, after which the whole was shaken with the aid of a Gyratory Shaker Model G2 (New Brunswick Scientific, USA). The immobilisation took place at 4 ⁇ C over a minimum of half an hour.
  • RDE Pt rotary disc electrode
  • a potential of 0.175 V against Ag reference was applied to the sensor.
  • a spiral-shaped Pt wire was used as auxiliary electrode.
  • the enzyme activity was determined with the aid of the above method.
  • the natural co- substrate oxygen
  • the synthetic electron acceptor benzoquinone The hydroquinone, which was formed during the catalytic cycle, was measured electrochemically using the rotary disc electrode (RDE) .
  • the regeneration of the benzoquinone from hydroquinone starts at the RDE at a certain potential (0.35 against Ag) and the resulting current is a measure for the enzyme activity.
  • Fig. 4 shows the effect on the measured current when the membrane according to the invention (see above) is introduced into the electro ⁇ chemical cell. As can be seen from Fig. 4, the current increases immediately following the introduction of the membrane. This effect occurs in the case of the Nuclepore membranes having initial pore diameters of 800 nm and 1000 nm, which have been indicated above.
  • Fig. 5 shows the electrochemical response of the system with respect to the addition of glucose.
  • the membrane was used as working electrode.
  • a potential of 0.175 V against Ag was applied to the membrane.
  • a platinum wire served as auxiliary electrode.
  • the amperometric response was determined in a stirred cell, which was filled with 1 ml of 0.1 M phos- phate buffer.
  • the increase in current strength (Fig. 5) was determined under an argon atmosphere and in the presence of 10 U/ml catalase.
  • the latter enzyme was added for the decomposition of any H 2 0 2 which may be produced enzymatically to accidental oxygen mediation.
  • the potential applied was, as stated, 0.175 against Ag reference, which is too low a potential to oxidise H 2 0 2 . Therefore, it can be stated that the enzyme transports its electrons directly to the conducting polymer.
  • the delimited space in the pores of the membrane, together with the amorphous structure of the polypyrrole, apparently brings the active centres of the enzyme mole- cules into close contact with the conducting polymer.
  • the response time is less than one minute, which can be regarded as rapid taking into account the geometry of the sensor.
  • the time which is needed to obtain a uniform glucose concentration throughout the entire solution must also be taken into account here; this time can be 10 seconds or more.
  • Glucose oxidase (E.C. 1.1.3-4) type II (25,000 U/g) from Aspergillus niger and catalase (E.C. 1.11.1.6, 2800
  • the used galvanostat was constructed at the University of Nijmegen, The Netherlands.
  • the current output of the galvanostat was monitored with a Fluke 5 digital multimeter. All electrochemical measurements were performed with an Autolab potentiostat controlled by an Olivetti M24 Personal Computer and General Purpose Electrochemical System (GPES)-software (Eco Chemie,
  • Agarose type VII was dissolved by boiling an appropriate amount (0.1 or 0.25 wt%) for 2 minutes in distilled water. Freshly made solutions, which were still hot, were used to make the latex membranes. A volume of agarose solution was mixed thoroughly with an equal volume of latex suspension. A 75 yl droplet was applied on a freshly sputtered platinum disk. After application, the electrode was put in the refrigerator overnight. The dried latex electrode was put in an oven at 333°K for 1 hour.
  • Latex electrodes were sealed with teflon tape in such a way as to leave only the latex surface for making contact with the polymerization medium.
  • An aqueous solution containing 0.9 % potassium chloride and 10 mM phosphate (PBS), together with 0.3 M pyrrole was used in the polymerization reaction.
  • the latex layer was put in the solution at least one minute before polymerization took place to allow the solution to penetrate the membrane sufficiently.
  • a constant current (20 mA/cm 2 ) was supplied to the cell for an appropriate time (see Table B) .
  • a platinum plate acted as counter electrode. When polymerization was finished the electrodes were rinsed with PBS.
  • Enzyme immobilization was achieved by agitating (Gyrotory
  • Enzyme activity was assayed with a three electrode cell containing 20 mL phosphate buffered saline (PBS), pH 7-5. 5 mM benzoquinone and 0.5 M glucose. Prior to use, the glucose solution was allowed to mutarotate for at least 24 hours.
  • the assay was performed with a platinum rotating disk electrode (6 mm diameter) equipped with an Electrocraft corporation model E550 motor and E552 speed control unit. The platinum working electrode was set at a potential of 0.350 V (Ag/AgCl reference) and was rotated at a speed of 2000 rpm. A platinum wire was used as auxiliary electrode.
  • the solution was flushed with argon before each experiment. During the assay argon was blanketed over the solution.
  • the actual assay was performed by monitoring the current output of the RDE while immersing a sample membrane into the solution.
  • the enzymatic activity of the various latex- polypyrrole membranes are indicated in Table B.
  • the enzyme membrane was placed as working electrode in a three electrode flow cell (Sparc Holland) .
  • a three electrode flow cell (Sparc Holland) .
  • To insulate the active surface of the membrane from the auxiliary electrode it was covered with a teflon spacer of 1 mm thickness. In the spacer a duct of approximately 0.15 cm 2 was left, allowing the membrane to make contact with the solution.
  • An Ag/AgCl electrode was used as reference electrode.
  • the base of the flow cell acted as auxiliary electrode (glassy carbon) .
  • Buffer solution was driven through the cell at a rate of 1.75 ml/minute (Watson Marlowe 101U peristaltic pump). The potential of the membrane was set at 0.350 V. When the background current had been diminished sufficiently, the buffer solution was replaced by the glucose solution and the current response was monitored, see Table C.
  • an enzyme electrode was constructed from the modified latex layer by treating it with glucose oxidase. Immobilization of glyucose oxidase inside the latex-polypyrrole pore structure was achieved by agitating a modified electrode in an aqueous solution (PBS) , containing the enzyme, for 4 hours and successively drying overnight. The immobilization procedure was conducted at a temperature of 277 K.
  • PBS aqueous solution
  • the enzyme electrodes were tested separately (viz. independent of the biosensor activity) for enzymatic activity by means of the Enzyme activity assay, described earlier.
  • the natural cosubstrate (oxygen) was replaced by the artificial electron acceptor benzoquinone.
  • Hydroquinone which is formed in the catalytic cycle, was measured electrochemically at a rotating disk electrode (RDE) .
  • RDE rotating disk electrode
  • the regeneration of benzoquinone from hydroquinone takes place at a fixed otential (0.35 vs. Ag/AgCl).
  • the resulting current is a measure of the enzymatic activity.
  • the raise in current as a result of the catalytic action of the enzyme is large enough to give a significant difference in slope of the current-time plot.
  • the boundary cases were, as visible in Table B, 400 and 500 mC/cm 2 for thin and thick layers respectively.
  • the current response of the different biosensors was tested by measuring various glucose concentrations with the individual enzyme treated latex-polypyrrole electrodes. Unless stated otherwise, the amperometric measurements were performed at a potential of 0.35 versus Ag/AgCl.
  • the polypyrrole-latex membranes were cast on platinum. Non-specific electrochemical glucose oxidation at the platinum surface could accidentally occur. Therefore, the electrodes were also tested for glucose sensitivity before they had been treated with glucose oxidase. No current response could be detected in this case. Therefore, non-specific oxidation of glucose at the electrode surface did not occur.
  • Thick layers (5 ⁇ m) consisting of 112 nm latex particles showed biosensor activity when treated with amounts of polypyrrole corresponding to 200-500 mC/cm 2 .
  • the same values apply for layers consisting of 220 nm particles. This is in concurrence with the data in Table B, where the enzymatic activity of these layers is listed.
  • significant differences in the absolute values of biosensor activity were measured when the two types of latex layers (viz. with 112 and 220 nm spheres resp.) were treated with various amounts of polypyrrole.
  • Fig. 11 the calibration curves are shown for enzyme electrodes composed of 112 nm diameter latex particles and amounts of polypyrrole corresponding to 200 and 400 mC/cm 2 respectively. Lower amounts of polypyrrole were not tested (see also Table B) .
  • Latex membranes with 220 nm particles showed somewhat different behaviour. Pyrrole deposition corresponding to 500 mC/cm 2 still yielded biosensor activity, while higher polypyrrole loading yielded inactive membranes (Fig. lib) .
  • the optimum in Fig. lib is for membranes with an amount of polypyrrole corresponding to a charge of 400 mC/cm 2 . Lower amounts of polypyrrole yielded less active and poorly performing membranes.
  • the optimum activity of sensors based on 5 ⁇ m membranes is approximately 60 +/- 10 nA/mM glucose (calculated from the linear area) . This can be compared to the activity of the 1 ⁇ m membranes (10 nA/mM glucose).
  • the enzyme loading of the thick membranes will be higher due to the availability of more conducting polymer surface, leading to an increased activity.
  • biosensors which displayed the highest activity were characterized further.
  • the dynamic range of these biosensors was tested by measuring a large range of glucose concentrations and the lifetime under quasi- continuous operation was evaluated.
  • Fig. 12a shows the response curve for a 1 ⁇ m latex layer with 220 nm particle size, covered with an amount of polypyrrole corresponding to 150 mC/cm 2 .
  • the current response to glucose was virtually linear up to 20 mM.
  • the various amounts of polypyrrole on 112 and 220 nm particles tested gave similar calibration lines as in Fig. 12a. This reproducable behaviour is lost upon going to 5 ⁇ m thick latex membranes.
  • Fig. 12b For the optimal latex-polypyrrole combinations a large difference in dynamic response is obtained for the different dimensions of the latex particles.
  • the dynamic range of sensors based on 220 nm particles is 60 mM, with a linear current response to glucose up to 10 mM (Fig. 12b, solid line). From Fig. 12b (dashed line) it can be seen that the calibration curve for the sensor with 112 nm latex beads reveals a dynamic range of about half this value.
  • the linearity on glucose concentration is less than 5 mM.
  • the response time of the biosensors depended on the amount of polypyrrole present, the thickness of the latex layer, and the particle size of the latex beads.
  • the various response times were evaluated by measuring 5 mM glucose and are summarized in Table C.
  • the response time is defined as the time needed to reach 95 % of the steady state current. It should be noted that this time is slightly dependent on glucose concentration. However, the difference in response time for the measurement of, e.g., 2 mM and 20 mM glucose was less than approximately 5 s.
  • a glucose concentration of 5 mM was introduced in the carrier stream and the increase in current was measured. In this way we were able to account for any deviations due to baseline drift and still the sensor lifetime was evaluated under continuous operation.
  • Fig. 13 the sensitivity of a latex-polypyrrole biosensor (220 nm, thick layer, 400 mC/cm 2 ) to 5 glucose is plotted as a function of time. The current response is not stable during the first days of measurement. Probably, in the beginning an amount of enzyme which is less firmly bound is slowly washed out. After 3 days, the sensor response remained the same for 10 days. This stability is sufficient for disposable applications.
  • the working potential (maximum 0.35 versus Ag/AgCl reference) of the latex-polypyrrole membrane electrodes was very low.
  • Polypyrrole is electroactive in its oxidized state and the charge carried by the conducting polymer can be cycled repetitively.
  • the confined space in the interspherical pores of the latex apparently brings the active centers of the enzyme molecules in close contact with the conducting polymer.
  • the adsorption process should play an important role in this by allowing the enzyme to penetrate the surface of polypyrrole. Applicant has found that drying after enzyme treatment of the membrane was essential for both the immobilization of enzyme and the direct electron transfer.
  • Water is likely to be a competing species with regard to adsorption on the polymer surface.
  • enzyme adsorption is favoured and the active centers of the enzyme can approach the conducting polymer sufficiently to make direct communication possible. Electrostatic interactions could also play an important role in the immobilization process.
  • Polypyrrole in its conducting state is a polycation.
  • Glucose oxidase in neutral solution has in its turn at least 10 negative charges on its surface (pi is 4.1). Therefore, electrostatic interactions may be very strong. Because of these features the electroactive sites on the conducting polymer could strongly interact with the enzyme thereby making direct electron transfer possible. (8) Conclusions
  • Amperometric glucose biosensors can be constructed from polypyrrole modified latex layers which are cast on a platinum electrode. Adsorption of glucose oxidase on the interspherical polypyrrole surface leads to immobilization of the redox protein without loss of enzymatic activity. the response to glucose of this biosensor probably is the result of direct electron transfer between glucose oxidase and polypyrrole. The confined space in the pores between the latex spheres, together with the surface morphology of polypyrrole make this possible. Electrostatic interactions contribute to this property. Measurements under argon and oxygen atmosphere show no significant difference in current response. The response to hydrogen peroxide at relatively low anodic potential (i.e. 0.10 V vs.
  • the principle of immobilizing redox enzymes by adsorption on a conducting polymer surface inside the pores of latex layers can be applied to many combinations of redox enzymes and conducting polymers.
  • the principle of sensor construction can be utilized to develop disposable sensors. The stability of the sensor is sufficient for this purpose.

Abstract

Electrode constituée d'une membrane traversée par des pores ouverts dont les parois possèdent un revêtement polymère électroconducteur auquel est liée une enzyme redox. Dans ce type d'électrodes, on peut obtenir un transfert direct d'électrons entre l'enzyme redox (par exemple la glucose-oxydase). Une telle électrode, qui est facilement produite, peut diversement s'appliquer, par exemple, à un capteur biologique ou à une installation de préparation de produits chimiques spécifiques. On peut employer, à titre de matériaux de départ pour ce type d'électrode, des particules de latex ainsi que des matériaux membraneux poreux disponibles dans le commerce. Les parois des pores de la membrane poreuse et les interstices des particules de latex sont munis respectivement d'une couche mince du polymère électroconducteur, ladite couche étant elle-même munie d'une enzyme redox appropriée.
PCT/NL1991/000263 1990-12-14 1991-12-13 Electrode pourvue d'un revetement polymere auquel est liee une enzyme redox WO1992010584A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
DE69104496T DE69104496T2 (de) 1990-12-14 1991-12-13 Elektrode, ausgestattet mit einer polymerbeschichtung mit einem daran gebundenen redoxenzym.
US08/075,509 US5422246A (en) 1990-12-14 1991-12-13 Electrode having a polymer coating with a redox enzyme bound thereto, the polymer coating being formed on the walls of pores extending through a porous membrane
EP92901729A EP0561966B1 (fr) 1990-12-14 1991-12-13 Electrode pourvue d'un revetement polymere auquel est liee une enzyme redox

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
NL9002764A NL9002764A (nl) 1990-12-14 1990-12-14 Elektrode, voorzien van een polymeerbekleding met een daaraan gebonden redox-enzym.
NL9002764 1990-12-14

Publications (1)

Publication Number Publication Date
WO1992010584A1 true WO1992010584A1 (fr) 1992-06-25

Family

ID=19858149

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/NL1991/000263 WO1992010584A1 (fr) 1990-12-14 1991-12-13 Electrode pourvue d'un revetement polymere auquel est liee une enzyme redox

Country Status (5)

Country Link
US (1) US5422246A (fr)
EP (1) EP0561966B1 (fr)
DE (1) DE69104496T2 (fr)
NL (1) NL9002764A (fr)
WO (1) WO1992010584A1 (fr)

Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004398A1 (fr) * 1994-08-01 1996-02-15 Medisense Inc. Electrodes et leur utilisation en analyse
WO1997043633A1 (fr) * 1996-05-17 1997-11-20 Encelle, Inc. Biocapteurs electrochimiques
US5922339A (en) * 1998-01-27 1999-07-13 Usala; Anton-Lewis Compositions and methods for biocompatible implants
USRE44695E1 (en) 2003-12-05 2014-01-07 Dexcom, Inc. Dual electrode system for a continuous analyte sensor
US8690775B2 (en) 2004-07-13 2014-04-08 Dexcom, Inc. Transcutaneous analyte sensor
US8840553B2 (en) 1998-04-30 2014-09-23 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US8845536B2 (en) 2003-08-01 2014-09-30 Dexcom, Inc. Transcutaneous analyte sensor
US8886272B2 (en) 2004-07-13 2014-11-11 Dexcom, Inc. Analyte sensor
US8915850B2 (en) 2005-11-01 2014-12-23 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US8915849B2 (en) 2003-08-01 2014-12-23 Dexcom, Inc. Transcutaneous analyte sensor
US8920319B2 (en) 2005-11-01 2014-12-30 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US8968198B2 (en) 2006-02-22 2015-03-03 Dexcom, Inc. Analyte sensor
US8974386B2 (en) 1998-04-30 2015-03-10 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US8986209B2 (en) 2003-08-01 2015-03-24 Dexcom, Inc. Transcutaneous analyte sensor
US9011332B2 (en) 2001-01-02 2015-04-21 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US9055901B2 (en) 2004-07-13 2015-06-16 Dexcom, Inc. Transcutaneous analyte sensor
US9066695B2 (en) 1998-04-30 2015-06-30 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US9078608B2 (en) 2005-03-10 2015-07-14 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US9078607B2 (en) 2005-11-01 2015-07-14 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US9131885B2 (en) 2009-07-02 2015-09-15 Dexcom, Inc. Analyte sensors and methods of manufacturing same
US9135402B2 (en) 2007-12-17 2015-09-15 Dexcom, Inc. Systems and methods for processing sensor data
US9149233B2 (en) 2007-12-17 2015-10-06 Dexcom, Inc. Systems and methods for processing sensor data
US9247900B2 (en) 2004-07-13 2016-02-02 Dexcom, Inc. Analyte sensor
US9351677B2 (en) 2009-07-02 2016-05-31 Dexcom, Inc. Analyte sensor with increased reference capacity
US9717449B2 (en) 2007-10-25 2017-08-01 Dexcom, Inc. Systems and methods for processing sensor data
US10022078B2 (en) 2004-07-13 2018-07-17 Dexcom, Inc. Analyte sensor
US10478108B2 (en) 1998-04-30 2019-11-19 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
GB2577493A (en) * 2018-09-25 2020-04-01 Sumitomo Chemical Co Wound dressing
US11399745B2 (en) 2006-10-04 2022-08-02 Dexcom, Inc. Dual electrode system for a continuous analyte sensor
US11633133B2 (en) 2003-12-05 2023-04-25 Dexcom, Inc. Dual electrode system for a continuous analyte sensor

Families Citing this family (92)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW279133B (fr) * 1990-12-13 1996-06-21 Elan Med Tech
AU1911795A (en) * 1994-02-09 1995-08-29 Abbott Laboratories Diagnostic flow cell device
IE72524B1 (en) * 1994-11-04 1997-04-23 Elan Med Tech Analyte-controlled liquid delivery device and analyte monitor
US6958245B2 (en) 1996-04-25 2005-10-25 Bioarray Solutions Ltd. Array cytometry
US7144119B2 (en) 1996-04-25 2006-12-05 Bioarray Solutions Ltd. System and method for programmable illumination pattern generation
CA2255599C (fr) * 1996-04-25 2006-09-05 Bioarray Solutions, Llc Assemblage electrocinetique de particules proches des surfaces regule par la lumiere
US6387707B1 (en) * 1996-04-25 2002-05-14 Bioarray Solutions Array Cytometry
US7041510B2 (en) * 1996-04-25 2006-05-09 Bioarray Solutions Ltd. System and method for programmable illumination pattern generation
ATE227844T1 (de) 1997-02-06 2002-11-15 Therasense Inc Kleinvolumiger sensor zur in-vitro bestimmung
US20030027126A1 (en) 1997-03-14 2003-02-06 Walt David R. Methods for detecting target analytes and enzymatic reactions
US7622294B2 (en) * 1997-03-14 2009-11-24 Trustees Of Tufts College Methods for detecting target analytes and enzymatic reactions
US6060327A (en) 1997-05-14 2000-05-09 Keensense, Inc. Molecular wire injection sensors
US7220550B2 (en) * 1997-05-14 2007-05-22 Keensense, Inc. Molecular wire injection sensors
US6699667B2 (en) 1997-05-14 2004-03-02 Keensense, Inc. Molecular wire injection sensors
JP2002505008A (ja) 1997-06-16 2002-02-12 エラン コーポレーション ピーエルシー 分析物のｉｎ ｖｉｖｏ測定のためのセンサーをキャリブレートし、試験する方法と、このような方法に用いるためのデバイス
US6949816B2 (en) 2003-04-21 2005-09-27 Motorola, Inc. Semiconductor component having first surface area for electrically coupling to a semiconductor chip and second surface area for electrically coupling to a substrate, and method of manufacturing same
US6251260B1 (en) 1998-08-24 2001-06-26 Therasense, Inc. Potentiometric sensors for analytic determination
US6591125B1 (en) 2000-06-27 2003-07-08 Therasense, Inc. Small volume in vitro analyte sensor with diffusible or non-leachable redox mediator
US6338790B1 (en) 1998-10-08 2002-01-15 Therasense, Inc. Small volume in vitro analyte sensor with diffusible or non-leachable redox mediator
EP1192269A2 (fr) 1999-06-18 2002-04-03 Therasense, Inc. CAPTEUR D'ANALYTE i IN VIVO /i A TRANSFERT DE MASSE LIMITE
US6616819B1 (en) 1999-11-04 2003-09-09 Therasense, Inc. Small volume in vitro analyte sensor and methods
KR100360774B1 (ko) 1999-12-27 2002-11-13 한국전자통신연구원 효소전극센서 및 그 제조방법
DE10022750A1 (de) * 2000-05-10 2001-11-22 Wolfgang Schuhmann Verfahren zur Immobilisierung von Erkennungskomponenten
US9709559B2 (en) 2000-06-21 2017-07-18 Bioarray Solutions, Ltd. Multianalyte molecular analysis using application-specific random particle arrays
US7892854B2 (en) 2000-06-21 2011-02-22 Bioarray Solutions, Ltd. Multianalyte molecular analysis using application-specific random particle arrays
US7057704B2 (en) * 2000-09-17 2006-06-06 Bioarray Solutions Ltd. System and method for programmable illumination pattern generation
US20030045005A1 (en) * 2000-10-17 2003-03-06 Michael Seul Light-controlled electrokinetic assembly of particles near surfaces
WO2002078512A2 (fr) 2001-04-02 2002-10-10 Therasense, Inc. Dispositif et procede de recherche de glucose dans le sang
US7262063B2 (en) 2001-06-21 2007-08-28 Bio Array Solutions, Ltd. Directed assembly of functional heterostructures
KR20040068122A (ko) 2001-10-15 2004-07-30 바이오어레이 솔루션스 리미티드 공동 검색과 효소-매개된 탐지에 의한 다형성 좌위의 다중분석
EP1461840A1 (fr) * 2001-12-11 2004-09-29 Powerzyme, Inc. Membranes biocompatibles stabilisees de copolymeres blocs et piles a combustible produites a l'aide de ces dernieres
US7813780B2 (en) * 2005-12-13 2010-10-12 Medtronic Minimed, Inc. Biosensors and methods for making and using them
US7041401B2 (en) * 2002-10-18 2006-05-09 University Of Iowa Research Foundation Magnetically modified electrodes containing at least one catalyst component that mediates a subatomic particle transfer process
US7526114B2 (en) 2002-11-15 2009-04-28 Bioarray Solutions Ltd. Analysis, secure access to, and transmission of array images
AU2003303597A1 (en) 2002-12-31 2004-07-29 Therasense, Inc. Continuous glucose monitoring system and methods of use
US7587287B2 (en) 2003-04-04 2009-09-08 Abbott Diabetes Care Inc. Method and system for transferring analyte test data
US8066639B2 (en) 2003-06-10 2011-11-29 Abbott Diabetes Care Inc. Glucose measuring device for use in personal area network
EP1502957B1 (fr) * 2003-07-11 2008-08-27 F. Hoffmann-La Roche Ag Membrane de diffusion à couler pour des capteur basés sur un enzyme
CA2473069C (fr) * 2003-07-11 2014-03-18 F. Hoffmann-La Roche Ag Membrane de diffusion moulable pour des applications de detection base sur un enzyme
WO2007120442A2 (fr) 2003-07-25 2007-10-25 Dexcom, Inc. Système d'électrode double pour capteur d'analyte continu
US8160669B2 (en) 2003-08-01 2012-04-17 Dexcom, Inc. Transcutaneous analyte sensor
WO2005029705A2 (fr) 2003-09-18 2005-03-31 Bioarray Solutions, Ltd. Codage numerique pour identification de sous-types des types de supports solides codes
CA2539824C (fr) 2003-09-22 2015-02-03 Xinwen Wang Polyelectrolyte immobilise en surface a plusieurs groupes fonctionnels pouvant se lier de maniere covalente a des biomolecules
WO2005040407A1 (fr) * 2003-10-24 2005-05-06 Bayer Healthcare Llc Biocapteur electrochimique enzymatique
AU2004286252A1 (en) 2003-10-28 2005-05-12 Bioarray Solutions Ltd. Optimization of gene expression analysis using immobilized capture probes
KR20050040714A (ko) * 2003-10-28 2005-05-03 티디케이가부시기가이샤 다공질 기능성막, 센서, 다공질 기능성막의 제조방법,다공질 금속막의 제조방법 및 센서의 제조방법
WO2005045060A2 (fr) 2003-10-29 2005-05-19 Bioarray Solutions, Ltd. Analyse multiplexee d'acide nucleique par fragmentation d'adn bicatenaire
US20090194427A1 (en) * 2003-12-31 2009-08-06 Shen-Kan Hsiung Using polypyrrole as the contrast ph detector to fabricate a whole solid-state ph sensing device
US20050147736A1 (en) * 2003-12-31 2005-07-07 Chung Yuan Christian University Using polypyrrole as the contrast pH detector to fabricate a whole solid-state pH sensing device
EP1718198A4 (fr) 2004-02-17 2008-06-04 Therasense Inc Procede et systeme de communication de donnees dans un systeme de controle et de gestion de glucose en continu
US7848889B2 (en) 2004-08-02 2010-12-07 Bioarray Solutions, Ltd. Automated analysis of multiplexed probe-target interaction patterns: pattern matching and allele identification
US8060174B2 (en) * 2005-04-15 2011-11-15 Dexcom, Inc. Analyte sensing biointerface
US8112240B2 (en) 2005-04-29 2012-02-07 Abbott Diabetes Care Inc. Method and apparatus for providing leak detection in data monitoring and management systems
US8486629B2 (en) 2005-06-01 2013-07-16 Bioarray Solutions, Ltd. Creation of functionalized microparticle libraries by oligonucleotide ligation or elongation
US8807350B2 (en) 2005-07-14 2014-08-19 Ben-Gurion University Of The Negev Research & Development Authority Composite membranes and methods for their preparation
US8709740B2 (en) * 2005-08-11 2014-04-29 The United States Of America, As Represented By The Secretary Of The Navy Control of protein activity using a conducting polymer
US7766829B2 (en) 2005-11-04 2010-08-03 Abbott Diabetes Care Inc. Method and system for providing basal profile modification in analyte monitoring and management systems
US7885698B2 (en) 2006-02-28 2011-02-08 Abbott Diabetes Care Inc. Method and system for providing continuous calibration of implantable analyte sensors
US8226891B2 (en) 2006-03-31 2012-07-24 Abbott Diabetes Care Inc. Analyte monitoring devices and methods therefor
US7620438B2 (en) 2006-03-31 2009-11-17 Abbott Diabetes Care Inc. Method and system for powering an electronic device
US7920907B2 (en) 2006-06-07 2011-04-05 Abbott Diabetes Care Inc. Analyte monitoring system and method
US8930203B2 (en) 2007-02-18 2015-01-06 Abbott Diabetes Care Inc. Multi-function analyte test device and methods therefor
US8732188B2 (en) 2007-02-18 2014-05-20 Abbott Diabetes Care Inc. Method and system for providing contextual based medication dosage determination
US8123686B2 (en) 2007-03-01 2012-02-28 Abbott Diabetes Care Inc. Method and apparatus for providing rolling data in communication systems
US8665091B2 (en) 2007-05-08 2014-03-04 Abbott Diabetes Care Inc. Method and device for determining elapsed sensor life
US8456301B2 (en) 2007-05-08 2013-06-04 Abbott Diabetes Care Inc. Analyte monitoring system and methods
US7928850B2 (en) 2007-05-08 2011-04-19 Abbott Diabetes Care Inc. Analyte monitoring system and methods
US8461985B2 (en) 2007-05-08 2013-06-11 Abbott Diabetes Care Inc. Analyte monitoring system and methods
US8702932B2 (en) 2007-08-30 2014-04-22 Pepex Biomedical, Inc. Electrochemical sensor and method for manufacturing
WO2009051901A2 (fr) * 2007-08-30 2009-04-23 Pepex Biomedical, Llc Capteur électrochimique et procédé de fabrication
WO2010056876A2 (fr) 2008-11-14 2010-05-20 Pepex Biomedical, Llc Fabrication d’un module de capteurs électrochimiques
US9445755B2 (en) 2008-11-14 2016-09-20 Pepex Biomedical, Llc Electrochemical sensor module
US8951377B2 (en) 2008-11-14 2015-02-10 Pepex Biomedical, Inc. Manufacturing electrochemical sensor module
US8103456B2 (en) 2009-01-29 2012-01-24 Abbott Diabetes Care Inc. Method and device for early signal attenuation detection using blood glucose measurements
US20100213057A1 (en) 2009-02-26 2010-08-26 Benjamin Feldman Self-Powered Analyte Sensor
WO2010127050A1 (fr) 2009-04-28 2010-11-04 Abbott Diabetes Care Inc. Détection d'erreur dans des données de répétition critiques dans un système de capteur sans fil
WO2010138856A1 (fr) 2009-05-29 2010-12-02 Abbott Diabetes Care Inc. Systèmes d'antenne de dispositif médical comportant des configurations d'antenne externe
US9314195B2 (en) 2009-08-31 2016-04-19 Abbott Diabetes Care Inc. Analyte signal processing device and methods
US8993331B2 (en) 2009-08-31 2015-03-31 Abbott Diabetes Care Inc. Analyte monitoring system and methods for managing power and noise
EP2482720A4 (fr) 2009-09-29 2014-04-23 Abbott Diabetes Care Inc Procédé et appareil de fourniture de fonction de notification dans des systèmes de surveillance de substance à analyser
US8660628B2 (en) 2009-12-21 2014-02-25 Medtronic Minimed, Inc. Analyte sensors comprising blended membrane compositions and methods for making and using them
US9504162B2 (en) 2011-05-20 2016-11-22 Pepex Biomedical, Inc. Manufacturing electrochemical sensor modules
EP2775918B1 (fr) 2011-11-07 2020-02-12 Abbott Diabetes Care Inc. Dispositif et procédés de contrôle de substance à analyser
AU2013300137A1 (en) * 2012-05-10 2015-01-15 The Regents Of The University Of California Wearable electrochemical sensors
US9968306B2 (en) 2012-09-17 2018-05-15 Abbott Diabetes Care Inc. Methods and apparatuses for providing adverse condition notification with enhanced wireless communication range in analyte monitoring systems
EP2900315B1 (fr) 2012-09-25 2017-09-06 Sorin CRM SAS Dispositif permettant de libérer électrochimiquement une composition de manière régulée
US11224367B2 (en) 2012-12-03 2022-01-18 Pepex Biomedical, Inc. Sensor module and method of using a sensor module
US10004434B1 (en) * 2013-03-15 2018-06-26 Georgetown University Microfluidic systems for electrochemical transdermal analyte sensing using a capillary-located electrode
CN105492902B (zh) * 2013-08-07 2020-07-24 爱科来株式会社 使用电化学式生物传感器的物质测量方法和测量装置
EP3152559B1 (fr) 2014-06-04 2020-12-02 Pepex Biomedical, Inc. Capteurs électrochimiques fabriqués à une technologie d'impression avancée
WO2016090189A1 (fr) 2014-12-03 2016-06-09 The Regents Of The University Of California Capteurs chimiques et biocapteurs non invasifs et portables
WO2017023931A1 (fr) 2015-08-03 2017-02-09 Georgetown University Appareil et procédé permettant l'administration d'un agent antimicrobien pendant un échantillonnage transdermique et procédé d'administration

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4582575A (en) * 1984-09-04 1986-04-15 Rockwell International Corporation Electrically conductive composites and method of preparation

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59164953A (ja) * 1983-03-10 1984-09-18 Fuji Electric Corp Res & Dev Ltd 固定化酵素膜およびその製造方法
US4589575A (en) * 1983-11-15 1986-05-20 Allan Rigberg Hygienic dispenser for wafers
JPS63218850A (ja) * 1987-03-09 1988-09-12 Meitetsuku:Kk 酵素電極およびその製造方法
US4891104A (en) * 1987-04-24 1990-01-02 Smithkline Diagnostics, Inc. Enzymatic electrode and electrode module and method of use
ES2052648T3 (es) * 1987-08-04 1994-07-16 Kao Corp Una membrana compuesta intercambiadora de cationes de polimeros conjugado y su proceso de preparacion.
FR2628746B1 (fr) * 1988-03-21 1992-03-27 Solvay Procede de preparation d'un latex a base de polypyrrole et utilisation de ce latex pour former un film conducteur
US5215682A (en) * 1988-03-21 1993-06-01 Solvay S.A. Process for preparing a latex based on polypyrrole and use of this latex to form a conducting film
US5200051A (en) * 1988-11-14 1993-04-06 I-Stat Corporation Wholly microfabricated biosensors and process for the manufacture and use thereof
US5202261A (en) * 1990-07-19 1993-04-13 Miles Inc. Conductive sensors and their use in diagnostic assays
US5250439A (en) * 1990-07-19 1993-10-05 Miles Inc. Use of conductive sensors in diagnostic assays

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4582575A (en) * 1984-09-04 1986-04-15 Rockwell International Corporation Electrically conductive composites and method of preparation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ANALYTICAL CHEMISTRY vol. 58, 1986, WASHINGTON DC USA pages 2979 - 2983; M. UMANA ET AL.: 'Protein-modified electrodes. The glucose / polypyrrole system.' cited in the application *
JOURNAL OF THE CHEMISTRY SOCIETY, CHEMICAL COMMUNICATIONS no. 14, 1989, USA pages 945 - 946; S.I. YABUKI ET AL.: 'Electro-conductive enzyme membrane.' *
PATENT ABSTRACTS OF JAPAN vol. 13, no. 12 (P-812)(3360) 12 January 1989 *
PATENT ABSTRACTS OF JAPAN vol. 9, no. 16 (P-329)(1739) 23 January 1985 & JP,A,59 164 953 ( FUJI DENKI SOUGOU KENKYUSHO K.K. ) 18 September 1984 *

Cited By (109)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004398A1 (fr) * 1994-08-01 1996-02-15 Medisense Inc. Electrodes et leur utilisation en analyse
US5849174A (en) * 1994-08-01 1998-12-15 Medisense, Inc. Electrodes and their use in analysis
WO1997043633A1 (fr) * 1996-05-17 1997-11-20 Encelle, Inc. Biocapteurs electrochimiques
US5922339A (en) * 1998-01-27 1999-07-13 Usala; Anton-Lewis Compositions and methods for biocompatible implants
US9011331B2 (en) 1998-04-30 2015-04-21 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US9072477B2 (en) 1998-04-30 2015-07-07 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US8840553B2 (en) 1998-04-30 2014-09-23 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US9066694B2 (en) 1998-04-30 2015-06-30 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US10478108B2 (en) 1998-04-30 2019-11-19 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US8880137B2 (en) 1998-04-30 2014-11-04 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US9066697B2 (en) 1998-04-30 2015-06-30 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US9042953B2 (en) 1998-04-30 2015-05-26 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US9014773B2 (en) 1998-04-30 2015-04-21 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US9066695B2 (en) 1998-04-30 2015-06-30 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US9326714B2 (en) 1998-04-30 2016-05-03 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US8974386B2 (en) 1998-04-30 2015-03-10 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US9011332B2 (en) 2001-01-02 2015-04-21 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US9498159B2 (en) 2001-01-02 2016-11-22 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US9610034B2 (en) 2001-01-02 2017-04-04 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US8986209B2 (en) 2003-08-01 2015-03-24 Dexcom, Inc. Transcutaneous analyte sensor
US8915849B2 (en) 2003-08-01 2014-12-23 Dexcom, Inc. Transcutaneous analyte sensor
US8845536B2 (en) 2003-08-01 2014-09-30 Dexcom, Inc. Transcutaneous analyte sensor
USRE44695E1 (en) 2003-12-05 2014-01-07 Dexcom, Inc. Dual electrode system for a continuous analyte sensor
US11633133B2 (en) 2003-12-05 2023-04-25 Dexcom, Inc. Dual electrode system for a continuous analyte sensor
US11045120B2 (en) 2004-07-13 2021-06-29 Dexcom, Inc. Analyte sensor
US10827956B2 (en) 2004-07-13 2020-11-10 Dexcom, Inc. Analyte sensor
US9044199B2 (en) 2004-07-13 2015-06-02 Dexcom, Inc. Transcutaneous analyte sensor
US8989833B2 (en) 2004-07-13 2015-03-24 Dexcom, Inc. Transcutaneous analyte sensor
US10314525B2 (en) 2004-07-13 2019-06-11 Dexcom, Inc. Analyte sensor
US11026605B1 (en) 2004-07-13 2021-06-08 Dexcom, Inc. Analyte sensor
US10993642B2 (en) 2004-07-13 2021-05-04 Dexcom, Inc. Analyte sensor
US10993641B2 (en) 2004-07-13 2021-05-04 Dexcom, Inc. Analyte sensor
US10980452B2 (en) 2004-07-13 2021-04-20 Dexcom, Inc. Analyte sensor
US10932700B2 (en) 2004-07-13 2021-03-02 Dexcom, Inc. Analyte sensor
US10918314B2 (en) 2004-07-13 2021-02-16 Dexcom, Inc. Analyte sensor
US10918315B2 (en) 2004-07-13 2021-02-16 Dexcom, Inc. Analyte sensor
US9247900B2 (en) 2004-07-13 2016-02-02 Dexcom, Inc. Analyte sensor
US10918313B2 (en) 2004-07-13 2021-02-16 Dexcom, Inc. Analyte sensor
US11064917B2 (en) 2004-07-13 2021-07-20 Dexcom, Inc. Analyte sensor
US9055901B2 (en) 2004-07-13 2015-06-16 Dexcom, Inc. Transcutaneous analyte sensor
US10813576B2 (en) 2004-07-13 2020-10-27 Dexcom, Inc. Analyte sensor
US10799158B2 (en) 2004-07-13 2020-10-13 Dexcom, Inc. Analyte sensor
US9414777B2 (en) 2004-07-13 2016-08-16 Dexcom, Inc. Transcutaneous analyte sensor
US11883164B2 (en) 2004-07-13 2024-01-30 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US10799159B2 (en) 2004-07-13 2020-10-13 Dexcom, Inc. Analyte sensor
US9603557B2 (en) 2004-07-13 2017-03-28 Dexcom, Inc. Transcutaneous analyte sensor
US8886272B2 (en) 2004-07-13 2014-11-11 Dexcom, Inc. Analyte sensor
US9668677B2 (en) 2004-07-13 2017-06-06 Dexcom, Inc. Analyte sensor
US10722152B2 (en) 2004-07-13 2020-07-28 Dexcom, Inc. Analyte sensor
US10709363B2 (en) 2004-07-13 2020-07-14 Dexcom, Inc. Analyte sensor
US10709362B2 (en) 2004-07-13 2020-07-14 Dexcom, Inc. Analyte sensor
US9833176B2 (en) 2004-07-13 2017-12-05 Dexcom, Inc. Transcutaneous analyte sensor
US10524703B2 (en) 2004-07-13 2020-01-07 Dexcom, Inc. Transcutaneous analyte sensor
US8858434B2 (en) 2004-07-13 2014-10-14 Dexcom, Inc. Transcutaneous analyte sensor
US8690775B2 (en) 2004-07-13 2014-04-08 Dexcom, Inc. Transcutaneous analyte sensor
US10022078B2 (en) 2004-07-13 2018-07-17 Dexcom, Inc. Analyte sensor
US9918668B2 (en) 2005-03-10 2018-03-20 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US9314196B2 (en) 2005-03-10 2016-04-19 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US11051726B2 (en) 2005-03-10 2021-07-06 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US9078608B2 (en) 2005-03-10 2015-07-14 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US11000213B2 (en) 2005-03-10 2021-05-11 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US10925524B2 (en) 2005-03-10 2021-02-23 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US10918316B2 (en) 2005-03-10 2021-02-16 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US9220449B2 (en) 2005-03-10 2015-12-29 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US10610136B2 (en) 2005-03-10 2020-04-07 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US10610137B2 (en) 2005-03-10 2020-04-07 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US10610135B2 (en) 2005-03-10 2020-04-07 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US10617336B2 (en) 2005-03-10 2020-04-14 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US10918318B2 (en) 2005-03-10 2021-02-16 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US10918317B2 (en) 2005-03-10 2021-02-16 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US10709364B2 (en) 2005-03-10 2020-07-14 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US10716498B2 (en) 2005-03-10 2020-07-21 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US10898114B2 (en) 2005-03-10 2021-01-26 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US10743801B2 (en) 2005-03-10 2020-08-18 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US10856787B2 (en) 2005-03-10 2020-12-08 Dexcom, Inc. System and methods for processing analyte sensor data for sensor calibration
US10813577B2 (en) 2005-06-21 2020-10-27 Dexcom, Inc. Analyte sensor
US11103165B2 (en) 2005-11-01 2021-08-31 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US9078607B2 (en) 2005-11-01 2015-07-14 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US11911151B1 (en) 2005-11-01 2024-02-27 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US8915850B2 (en) 2005-11-01 2014-12-23 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US8920319B2 (en) 2005-11-01 2014-12-30 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US11399748B2 (en) 2005-11-01 2022-08-02 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US10952652B2 (en) 2005-11-01 2021-03-23 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US10201301B2 (en) 2005-11-01 2019-02-12 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US11363975B2 (en) 2005-11-01 2022-06-21 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US11272867B2 (en) 2005-11-01 2022-03-15 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US9326716B2 (en) 2005-11-01 2016-05-03 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US10231654B2 (en) 2005-11-01 2019-03-19 Abbott Diabetes Care Inc. Analyte monitoring device and methods of use
US8968198B2 (en) 2006-02-22 2015-03-03 Dexcom, Inc. Analyte sensor
US9724028B2 (en) 2006-02-22 2017-08-08 Dexcom, Inc. Analyte sensor
US11399745B2 (en) 2006-10-04 2022-08-02 Dexcom, Inc. Dual electrode system for a continuous analyte sensor
US11272869B2 (en) 2007-10-25 2022-03-15 Dexcom, Inc. Systems and methods for processing sensor data
US9717449B2 (en) 2007-10-25 2017-08-01 Dexcom, Inc. Systems and methods for processing sensor data
US10182751B2 (en) 2007-10-25 2019-01-22 Dexcom, Inc. Systems and methods for processing sensor data
US9901307B2 (en) 2007-12-17 2018-02-27 Dexcom, Inc. Systems and methods for processing sensor data
US9149234B2 (en) 2007-12-17 2015-10-06 Dexcom, Inc. Systems and methods for processing sensor data
US10506982B2 (en) 2007-12-17 2019-12-17 Dexcom, Inc. Systems and methods for processing sensor data
US9839395B2 (en) 2007-12-17 2017-12-12 Dexcom, Inc. Systems and methods for processing sensor data
US10827980B2 (en) 2007-12-17 2020-11-10 Dexcom, Inc. Systems and methods for processing sensor data
US9135402B2 (en) 2007-12-17 2015-09-15 Dexcom, Inc. Systems and methods for processing sensor data
US9149233B2 (en) 2007-12-17 2015-10-06 Dexcom, Inc. Systems and methods for processing sensor data
US9339238B2 (en) 2007-12-17 2016-05-17 Dexcom, Inc. Systems and methods for processing sensor data
US11342058B2 (en) 2007-12-17 2022-05-24 Dexcom, Inc. Systems and methods for processing sensor data
US9237864B2 (en) 2009-07-02 2016-01-19 Dexcom, Inc. Analyte sensors and methods of manufacturing same
US9131885B2 (en) 2009-07-02 2015-09-15 Dexcom, Inc. Analyte sensors and methods of manufacturing same
US9763608B2 (en) 2009-07-02 2017-09-19 Dexcom, Inc. Analyte sensors and methods of manufacturing same
US9517025B2 (en) 2009-07-02 2016-12-13 Dexcom, Inc. Analyte sensor with increased reference capacity
US9351677B2 (en) 2009-07-02 2016-05-31 Dexcom, Inc. Analyte sensor with increased reference capacity
GB2577493A (en) * 2018-09-25 2020-04-01 Sumitomo Chemical Co Wound dressing

Also Published As

Publication number Publication date
DE69104496T2 (de) 1995-02-23
DE69104496D1 (de) 1994-11-10
NL9002764A (nl) 1992-07-01
EP0561966B1 (fr) 1994-10-05
EP0561966A1 (fr) 1993-09-29
US5422246A (en) 1995-06-06

Similar Documents

Publication Publication Date Title
US5422246A (en) Electrode having a polymer coating with a redox enzyme bound thereto, the polymer coating being formed on the walls of pores extending through a porous membrane
Pantano et al. Enzyme‐modified microelectrodes for in vivo neurochemical measurements
Koopal et al. Glucose sensor utilizing polypyrrole incorporated in tract-etch membranes as the mediator
US5264092A (en) Redox polymer modified electrode for the electrochemical regeneration of coenzyme
US5231028A (en) Immobilized enzyme electrodes
De Benedetto et al. One-step fabrication of a bienzyme glucose sensor based on glucose oxidase and peroxidase immobilized onto a poly (pyrrole) modified glassy carbon electrode
RU1801119C (ru) Ферментный электрод, амперометрически реагирующий на присутствие субстрата соответствующего фермента в анализируемом образце
Khan et al. Molecular interface for an activity controlled enzyme electrode and its application for the determination of fructose
Koopal et al. Third-generation glucose biosensor incorporated in a conducting printing ink
JP2003510570A (ja) 連続アナライトモニタリング用小型バイオセンサー
Coche-Guérente et al. Electrochemical immobilization of glucose oxidase in poly (amphiphilic pyrrole) films and its application to the preparation of an amperometric glucose sensor
ITMI951441A1 (it) Nuovi biosensori elettrochimici basati su nuovi trasduttori compositi
Koopal et al. Third-generation amperometric biosensor for glucose. Polypyrrole deposited within a matrix of uniform latex particles as mediator
Dempsey et al. Electropolymerised o-phenylenediamine film as means of immobilising lactate oxidase for a L-lactate biosensor
Khan et al. Platinization of shapable electroconductive polymer film for an improved glucose sensor
Nakabayashi et al. Amperometric glucose sensors fabricated by electrochemical polymerization of phenols on carbon paste electrodes containing ferrocene as an electron transfer mediator
JPS6239900B2 (fr)
Cosnier et al. A composite clay glucose biosensor based on an electrically connected HRP
Bardeletti et al. Amperometric enzyme electrodes for substrate and enzyme activity determinations
Jaffari et al. Novel hexacyanoferrate (III)-modified carbon electrodes: application in miniaturized biosensors with potential for in vivo glucose sensing
Markas Prototype, solid-phase, glucose biosensor
Qian et al. Polyelectrolyte-stabilized glucose biosensor based on woodceramics as electrode
Khan et al. Amperometric biosensor with PQQ enzyme immobilized in a mediator-containing polypyrrole matrix
WO1988008447A1 (fr) Procede amperometrique servant a la determination quantitative de dinucleotide de 1,4-dihydronicotinamide-adenine en solution
Aizawa et al. Electronically modulated biological functions of molecular interfaced enzymes and living cells

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU MC NL SE

WWE Wipo information: entry into national phase

Ref document number: 1992901729

Country of ref document: EP

Ref document number: 08075509

Country of ref document: US

WWP Wipo information: published in national office

Ref document number: 1992901729

Country of ref document: EP

WWG Wipo information: grant in national office

Ref document number: 1992901729

Country of ref document: EP